Establishment And Evaluation Of Glucose Modified Artesunate And Ligustrazine Hydrochloride Co-loaded Liposomes

Objective:Plasmodium falciparum is the most prevalent malaria parasite in sub-Saharan Africa,and it is also the main cause of the high mortality rate of Plasmodium infection.According to statistics,in 2016,nearly 92%of malaria-infected persons in sub-Saharan regions died from Plasmodium falciparum infection.Among them,15%～40%of patients died of Cerebral malaria（CM）.However,currently used in the treatment of cerebral malaria lacks brain targeting,and it is difficult to penetrate the Blood-Brain Barrier（BBB）.Based on this,this topic used glucose as the target and GLUT1 as the target protein（PDBID:4PYP）.Synthesize cholesterol-undecanoic acid-glucose conjugate with the ability to cross the blood-brain barrier.Preparation of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes.And then optimize its single-factor prescription and investigate its pharmacological properties.In vivo antimalarial activity was studied on glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes.Methods:1.Virtual screening of targeted materialsScreening of target molecules with the strongest effect on GLUT1 through molecular docking screening,then through molecular dynamics simulation and binding free energy to further verify the selected targeted molecules.2.Synthesis and characterization of cholesterol-undecanoic acid-glucose conjugateCholesterol was used as raw material to generate cholesterol-11 bromo-undecanoate through dehydration condensation reaction.Cholesterol-11 bromo-undecanoate and arbutin are condensed to obtain cholesterol-undecanoic acid-glucose conjugate.The structure was confirmed by mass spectrometry（MS）,infrared absorption spectroscopy（IR）,proton nuclear magnetic resonance spectroscopy,and carbon spectroscopy(¹H-NMR,¹³C-NMR).3.Preparation and formulation optimization of glucose-modified artesunate andligustrazine hydrochloride co-loaded liposomesPreparation of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes by thin film dispersion method.Encapsulation efficiency（EE）,particle size（size）and Polydispersity Index（PDI）are evaluation indicators to investigate the effects of phospholipid type,phospholipid dosage,cholesterol dosage,DSPE-m PEG2000 dosage and cholesterol-undecanoic acid-glucose conjugate dosage on liposomes.Then through orthogonal experiments to obtain a better prescription,and then optimize the preparation process.The surface morphology of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes was observed by transmission electron microscopy（TEM）.A laser particle size analyzer was used to investigate the particle size and polydispersity coefficient of liposomes.The encapsulation efficiency of liposomes was determined by ultrafiltration and centrifugation.4.Study on drug release of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomesHigh performance liquid chromatography（HPLC）was used to establish an in vitro analytical method for artesunate（ATS）and ligustrazine hydrochloride（TMP）.Next,the drug release study of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes.5.Evaluation of in vivo efficacy of glucose-modified artesunate and ligustrazinehydrochloride co-loaded liposomesC57 mice with malaria was utilized to evaluate efficacy of liposomes.Model group and blank liposome group was used as the negative control group.Original solution injection group（artesunate and ligustrazine hydrochloride injection）,original solution nose drops group,nose drops liposome group without targeting materials,liposome injection group with targeting materials and nose drops with targeting materials were set as experimental control groups.The infection rate,the rate of negative conversion,and the rate of re-ignition were evaluated as indicators,and the antimalarial activity in vivo of different administration methods and different dosage forms was investigated by Pearson’s four-day inhibition method.Use IVIS Lumina III small animal live imaging system to quantitatively analyze mouse brain fluorescence.Results:1.Virtual screening of targeted materialsThe 11-carbon optimal aliphatic chain was screened out by docking aliphatic chains of different lengths,and its docking score with GLUT1 was-6.93.Molecular dynamics simulation found that the combination of cholesterol-undecanoic acid-glucose conjugate with GLUT1 has little effect on GLUT1 protein and can form a stable hydrogen bond with GLUT1.The binding free energy found that the binding free energy of cholesterol-undecanoic acid-glucose conjugate with 4PYP protein is lower than that of the protein ligand in the PDB protein library.2.Synthesis and characterization of cholesterol-undecanoic acid-glucose conjugateThe conjugate is a white amorphous powder,soluble in ethanol and tetrahydrofuran;its ESI-MS,infrared spectrum,1D-NMR and 2D NMR spectrum characteristics are consistent with the structure of the target compound,indicating the successful synthesis of cholesterol-undecanoic acid-Glucose conjugate.3.Preparation and formulation optimization of glucose-modified artesunate andligustrazine hydrochloride co-loaded liposomesThe phospholipid type was determined to be 96%phospholipids through single factor experiments.The optimal liposome formulation is determined by orthogonal experiment:300mg phospholipids,25mg cholesterol,15mg cholesterol-undecanoic acid-glucose conjugate and 25 mg DSPE-m PEG2000.Under this prescription,the liposomes co-loaded with glucose-modified artesunate and ligustrazine hydrochloride have a bright appearance,and transmission electron microscopy shows that the liposomes are nearly spherical in shape.The average particle size of the glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes is 91.03±0.5nm;the encapsulation efficiency of artesunate and ligustrazine hydrochloride measured by ultrafiltration method are 93.10±0.50%and36.28±0.52%respectively.4.Study on drug release of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomesAn HPLC method was established for the determination of artesunate ligustrazine hydrochloride and the in vitro analysis of its preparations.After verification of the analytical method,artesunate has a good linear relationship within the concentration range of 5～2000μg/ml;ligustrazine hydrochloride has a good linear relationship within the concentration range of 5～100 0μg/ml.The precision,recovery rate,stability,and repeatability of the method all meet the methodological requirements.The in vitro release behavior of glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes showed that Liposomes have good sustained-release characteristics.The release amounts of artesunate and ligustrazine hydrochloride reached 82%and 61%,respectively,within 12 hours,and there was no obvious burst release phenomenon.5.Evaluation of in vivo efficacy of glucose-modified artesunate and ligustrazinehydrochloride co-loaded liposomesThe blank liposome group and the model group had no significant difference in the infection rate of Plasmodium（P>0.05）,indicating that the blank liposome group had no inhibitory effect on the Plasmodium.The results of the infection rate,negative conversion rate,and re-ignition rate of each preparation group showed that the antimalarial order of each administration group was the nasal liposome group with target>the liposome injection group with the target>the original liquid injection group>the no target drop nasal liposome group>original liquid nasal drip group.Conclusion:1.Molecular docking and molecular dynamics simulation found that the targeted material can form a stable interaction with GLUT1.2.The cholesterol-undecanoic acid-glucose conjugate was successfully synthesized,and its structure was confirmed by MS,IR,1H-NMR and 13C-NMR.3.Glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes prepared by the formulation optimized by the orthogonal experiment.The liposome’s morphology,particle size and distribution,in vitro release,encapsulation rate and other indicators all meet the expected requirements.4.The in vivo antimalarial pharmacodynamic test showed that the liposomes co-loaded with glucose-modified artesunate and ligustrazine hydrochloride had obvious antimalarial effects.Its anti-malarial effect is better than other administration groups.In vivo imaging analysis found that the glucose-modified artesunate and ligustrazine hydrochloride co-loaded liposomes have good brain targeting.